Biology, Medicine, & Natural Product Chemistry ISSN 2089-6514 (paper) Volume 13, Number 1, April 2024 | Pages: 109-119 | DOI: 10.14421/biomedich.2024.131.109-119 ISSN 2540-9328 (online) Comparative Effects of Ripe and Unripe Lime (Citrus aurantifolia) on Spermatozoa and Gonadosomatic Index in Matured Male Wistar Rats Obioma Ogbonnaya1, Azubuike Nwankwo2, Daniel Chidi Ifenkwe3, Chikera Samuel Ibe4, Ekele Ikpegbu4,5, Osah Martins Onwuka6,* 1College of Agriculture, Gregory University Uturu, Abia state, Nigeria. 2Department of Human Physiology, Faculty of Basic Medical and Health Sciences, Abia State University Uturu, Abia State, Nigeria. 3Department of Physiology and Pharmacology, College of Veterinary Medicine, Michael Okpara University of Agriculture, Umudike, Nigeria. 4Department of Veterinary Anatomy, College of Veterinary Medicine, Michael Okpara University of Agriculture Umudike, Abia State, Nigeria. 5Department of Comparative Pathology, Cummings School of Veterinary Medicine, Tufts University, United State of America. 6Department of Human Physiology, Gregory University Uturu, Abia State, Nigeria. Corresponding author* osahmartinz@gmail.com Manuscript received: 17 January, 2024. Revision accepted: 08 May, 2024. Published: 16 May, 2024. Abstract To ascertain the comparative effects of ripe and unripe Lime (Citrus aurantifolia) on spermatozoa and gonadosomatic index evaluation in matured male Wistar rats; exploring the idea that both ripe and unripe Lime (Citrus aurantifolia) might or might not positively affect semen quality, crucial for male fertility. Twenty-eight (28) sexually mature male Wister rats, aged 9–10 weeks and weighing between 211.50g and 217.00g, were divided equally into seven groups (1 to 7); with Group 1 serving as the control and Groups 2 to 7 receiving 25%, 50%, and 75% concentrations of ripe lime juice (RLJ) and unripe lime juice (ULJ) respectively. The findings indicated that ULJ had a higher concentration (0.1mg/ml) compared to ripe lime RLJ at 0.08mg/ml, although both had approximately the same LD50 value of 1581.138mg/kg. RLJ, at different concentrations, adversely impacted the reproductive performance of rats, leading to decreased progressive motility, livability, sperm count, testicular size, and sexual drive. However, ULJ did not exhibit these effects. A 75% concentration of RLJ showed anti-prostatic activity, causing a reduction in prostate size, which was more pronounced than that of the same ULJ concentration. Importantly, both RLJ and ULJ did not have a significant impact on the sizes of the liver, spleen, heart, kidneys, and lungs, with these visceral organs maintaining normal sizes comparable to the control group (statistically, p>0.05).The findings suggest that RLJ or ULJ consumption, particularly at the highest concentration, may lead to alterations in reproductive performance, hence such consumption should be discouraged. Keywords: Citrus aurantifolia; Lime, Fertility; Male reproduction; spermatozoa; Testis. INTRODUCTION Infertility is a phenomenon associated with offspring production (Rahayu and Hanizar, 2021). Globally, the incidence rate is approximately 30%, with males accounting for about 50% prevalence (Orieke et al., 2019). In Nigeria, male infertility is reported to contribute to 20-50% of all infertility cases across different regions (Eze, 2012). The worldwide increase in male infertility cases has been linked to a decline in semen quality (Abarikwu, 2013). Infertility can result from factors such as low sperm count, inadequate sperm motility, abnormal morphological structures, or a combination of these issues (Kumar and Singh, 2015). Another significant contributing factor is the decline in or inappropriate secretion of male sex hormones (Aprioku and Obianime, 2014; Majzoub and Agarwal, 2018). A normal sperm should have a concentration of at least 15 million/ml of ejaculate, with 72% motility indicating progressive movement and 94% normal morphology (World Health Organization, 2010). Abnormalities in sperm, particularly in concentration and motility, have been identified as major causes of male infertility in various studies (Sharma, 2017; Wdowiak, 2019; Babakhanzadeh, 2020). Other factors affecting sperm quality include exposure to radiation (Gorpinchenko, 2014; Kesari et al., 2018), nutrition (Martinez-Soto, 2016; Salas-Huetos et al., 2018), lifestyle choices (Ilacqua, 2018), age (Silea, 2019), psychological factors (Janevic, 2014), and deletions in the Y chromosome (Colaco and Modi, 2018). Several plants have a historical reputation for influencing male reproductive functions. Some of these plants can impact spermatogenesis at the testicular level or alter the body's hormone profile. The use of fruits like Citrus aurantifolia in the general diet is gaining popularity globally due to its nutritional and health https://doi.org/10.14421/biomedich.2024.131.109-119 mailto:osahmartinz@gmail.com 110 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 109-119 benefits (Nnenne et al., 2020). Commonly known as lime or bitter orange in Nigeria, this fruit belongs to the Rutaceae family. Lime is recognized for its rich content of phytochemicals, antioxidants, vitamins, minerals, and dietary fiber, which are known to reduce the risk of various health issues, as utilized in ethno-medicine (Septembre-Malaterre et al., 2018). It is considered a promising fruit with health-promoting qualities and is widely consumed (Adegoke et al., 2017). Lime, a sour, round, and bright green citrus fruit, is extensively used in West Africa, particularly in Nigeria, for various purposes such as food, refreshing drinks, desserts, and seasoning (Enejoh et al., 2015). Known for its health benefits, including immune system enhancement (Kim, 2023) and weight loss due to its hypolipidemic property (Enejoh et al., 2015), lime has become popular among young men for its perceived impact on weight management (Saini et al., 2022). This research aims to investigate the potential effects of lime consumption on the male reproductive system, including semen parameters, libido, hormonal levels, and testicular weight, using a rat model. Given lime's significant presence in herbal preparations in Nigeria (Ayinde et al., 2012; Enejoh et al., 2015), the findings will contribute to understanding how lime extract may influence physiological parameters and fertility functions in male Wister rats. This information is crucial for researchers, ethno-medicinal healers, and end-users, providing insights into the appropriate use of lime, especially among individuals of reproductive age in rural areas. MATERIAL AND METHODS Study Location Fresh lime fruits were collected and identified at the mini-market of Abia State University, Uturu Campus. The extraction of lime juice and the animal studies, including semen evaluation, were conducted in the Department of Physiology and Pharmacology at the College of Veterinary Medicine (CVM), Michael Okpara University of Agriculture, Umudike (MOUAU), where study approval was obtained. Experimental Animals and Management A total of 28 sexually matured male Wister rats, aged 9– 10 weeks and weighing between 211–217g, were obtained from the Department of Veterinary Physiology and Pharmacology animal house at the College of Veterinary Medicine (CVM), MOUAU. The animals underwent a 14-day acclimatization period in the animal laboratory, during which they were dewormed using Paraquantel (at 10mg/body weight), and their physiological parameters were measured and recorded. Under standard laboratory conditions with a 12-hour light–dark cycle at a room temperature of 25 ± 2oC, the rats were provided with a standard diet (pelleted Chukun growers feed) and had access to water ad libitum. Regular weighing was conducted using a sensitive electronic scale (digital electronic balance, Citizen Scales [1] PVT Ltd., South Patel Nagar, New Delhi, sensitivity: 0.01 g) before the start of the study and on a weekly basis. Preparation of lime juice extract Fresh lime fruits, both ripe and unripe, were washed, weighed, and halved. The juice from each half was expressed into a glass beaker. After filtration using muslin, the filtrate was collected into labeled bottles. The lime juice extract was considered 100%, and dilutions of 25%, 50%, and 75% were prepared by adding distilled water using volumetric flasks. These different concentrations were orally administered to the animals in separate groups, each receiving a dose of 5ml/kg of the respective lime juice concentration (Khan et al., 2010). The percentage yield of the juice extract was calculated using the formula: % yield = Weight of lime juice extracted Weight of lime fruit used × 100 (Soffwora, 1993) Acute toxicity study of the lime extract The Lorke method (1983) was employed in this study, which involved a total of 35 matured mice conducted in two phases. In the first phase, groups A-C, each comprising 5 mice, received oral doses of 10 mg/kg, 100 mg/kg, and 1000 mg/kg of ripe lime juice (RLJ) extract, respectively. In the second phase, groups D-F, also with 5 mice in each group, were orally administered doses of 1600 mg/kg, 2900 mg/kg, and 5000 mg/kg of unripe lime juice (ULJ) extract, respectively. Additionally, a control group (group G) consisting of 5 mice was given 5ml/kg of distilled water. All mice had unrestricted access to food and water ad libitum. The animals were observed for signs of toxicity and mortality over 72 hours post- administration and for an additional 14 days for delayed toxicity. The median lethal dose (LD50) of the lime juice extracts, both ripe and unripe, was calculated using the formula recommended by Khan et al. (2013). LD50 = √(least dose with mortality × Highest dose without mortality) Experimental Design The 28 male Wistar rats were evenly divided into 7 groups, including a control group, as well as groups receiving ripe lime juice (RLJ) and unripe lime juice (ULJ) at concentrations of 25%, 50%, and 75% from the stock solution (Table 1). Ogbonnaya et al. – Comparative Effects of Ripe and Unripe Lime (Citrus aurantifolia) … 111 Table 1. Experimental design. Group 1 Normal male rat (5ml/kg D/W) 2 RLJ at LD25% stock 3 RLJ at LD50% stock 4 RLJ at LD75% stock 5 ULJ at LD25% stock 6 ULJ at LD50% stock 7 ULJ at LD75% stock NOTE: RLJ: Ripe lime juice; ULJ: Unripe lime juice; LD: Lethal dose; D/W: Distilled water. The animals were dosed based on their body weight using a gastric tube (gavage) over a period of 28 days, during which continuous monitoring took place. After the dosing period, euthanasia was carried out by placing each rat in a small transparent bucket with a cover, containing cotton wool soaked in mild chloroform. The epididymis was then harvested, and sperm samples were collected from the caudal portion of the epididymis. Smears were prepared on preheated glass slides for subsequent evaluation. Male Fertility Study Libido (sex drive) evaluation Libido in the rats was assessed through physical observation, focusing on their reactions and sexual behavior when introduced to a female rat in estrus. Sexual behavioral reactions, including general grooming, sniffing, mounting, and thrusting on the female rats, were noted and recorded according to a scoring pattern described by Chibundu (2013) (Table 2). Scores were allocated based on these observed reactions. Table 2. Libido grading for male rats. Sexual behavior Score Grading Vigorous grooming, Sniffs and attempt to mount 5 Very high libido Grooms, sniffs but no attempt to mount 4 High libido Sniffs only 3 Moderate Grooms only 2 Low Does not pay attention to the female rat 1 Poor libido Source: Chibundu (2005). Sperm colour and consistency The color and consistency of the sperm samples were assessed through macroscopic observation and recorded. The evaluation followed the consistency scale (1-4) as defined by Chibundu (2013), where scores 1 and 2 represented milky white and creamy white, respectively. The consistency scale utilized included scores 1 (watery), 2 (slightly thick), 3 (thick), and 4 (very thick) for assessing the thickness of the sperm samples. Sperm motility The methodology outlined by El-Sherbiny (1987) was followed for evaluating sperm samples collected through epididymal washings in each treatment group. Progressive motile sperm cells were assessed immediately after collection. A smear of one drop of the sperm sample was created on a preheated glass slide and examined under light microscopy at low magnifications (×10 and ×40). The assessment was conducted subjectively and scored as a percentage. Only sperm cells demonstrating straight-forward movement were considered in the motility count, while those moving in circles, backward, or displaying pendulum-like movement were excluded. Individual sperm motility was scored according to the following scale: 1 (vigorously progressive), 2 (progressive), 3 (cycling movement), and 4 (stationary movement). Sperm viability (live proportion) The viability of sperm cells was determined by staining a drop of the collected sperm sample with Eosin-Nigrosin stain. The stained glass slide was allowed to air-dry for 30 seconds, followed by fixation with ethanol. The slide was then examined under a light microscope at a magnification of X100 (oil immersion). Using a hand- held mechanical stopwatch counter, the proportion of viable sperm cells was counted. A total of 300 sperm cells were counted, and the number of viable cells was expressed as a percentage of the total count. Viable sperm cells, which remain unstained, were differentiated from dead cells, which picked up the stain, following the method described by El-Sherbiny (1987). Sperm concentration The sperm concentration was determined using a haemocytometer following the method described by Ukar et al. (2016). A dilution of 1:200 was created using a red blood cell pipette. In this procedure, a 1% buffered formalin solution was employed as the semen diluting fluid to immobilize the sperm cells. The haemocytometer was loaded with a drop of the sperm solution, allowed to settle for 2 minutes on a wet paper (to facilitate sperm cell settling), and then examined under the microscope at ×40 magnification. The formular; Sperm concentration per ml = No. of cells counted × Dilution Factor × 0.04 × 106 (Egbuka, 1995) was used to determine sperm concentration. Abnormal sperm proportion The percentage of abnormal sperm proportion was assessed following the method outlined by El-Sherbiny (1987). A drop of the sperm sample was stained with Eosin-Nigrosin stain, and the resulting mixture was smeared on a glass slide. Under a lower magnification of ×40, the slide was examined to identify primary and secondary abnormal sperm cells. The percentages of differential abnormalities, including head, mid-piece, and 112 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 109-119 tail abnormalities, were determined according to the criteria specified by El-Sherbiny (1987). Statistical Analysis All collected data underwent statistical analysis, and the test values were presented as Mean ± SEM (Standard Error of Mean). Statistical differences among the means of different groups were analyzed using one-way analysis of variance (ANOVA), followed by the LSD test for mean separation. The level of significance was set at p < 0.05 compared to the control group. Values at p < 0.05 were considered statistically significant. RESULTS Comparing juice percentage yield of ripe and unripe lime juice The RLJ yielded (72.50%), more than the ULJ (61.25%) (Figure 1). Figure 1. Comparing juice percentage yield of ripe and unripe lime juice. Comparing juice concentration yield of unripe lime juice On the contrary, the ULJ was more concentrated (100mg/ml; p<0.05) than the RLJ (80mg/ml) (Figure 2). Figure 2. Comparing juice concentration yield of unripe lime juice. Acute toxicity testing of ripe and unripe lime juice The lethality results indicated that, after 72 hours post- administration of both ripe lime juice (RLJ) and unripe lime juice (ULJ) to rats at high (5000mg/kg) and low (500mg/kg) doses, no deaths or signs of toxicity were recorded in the low dose (500mg/kg) groups for both RLJ and ULJ. However, in the 5000mg/kg dose groups, 100% mortality was observed in RLJ, and 75% mortality was recorded in ULJ. The calculated LD50 was 1581.138mg/kg, falling below the 2000mg/kg threshold, indicating significant lethality in both RLJ and ULJ. Based on these findings, convenient concentration gradients of 25%, 50%, and 75% reconstitution were prepared and utilized in the study for both RLJ and ULJ. Effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa assessment of male wistar rats Table 3 illustrates the significant effects of both ripe lime juice (RLJ) and unripe lime juice (ULJ) administration on the spermatozoa qualities (color, pH, progressive motility, livability, and count) of Wistar male rats. However, the thickness/consistency of the epididymal reserve was not affected. The color of the epididymal reserve in male Wistar rats administered ULJ at 25% and 50% concentrations shifted from milky white (normal color) to creamy white. In contrast, the color of the epididymal reserve in those administered RLJ at various concentrations remained unchanged and retained a milky white appearance similar to that of normal rats. The pH of the epididymal reserve in male Wistar rats administered graded concentrations of RLJ (25%, 50%, and 75%) and 25% concentration of ULJ fell within the normal pH range (7.00±0.00) of rat semen (pH 6.99±0.00). However, the pH of the epididymal reserve was significantly lower (p < 0.05) at 6.98±0.01 in the group of Wistar rats administered the highest concentration (75%) of ULJ compared to the normal pH. The results of spermatozoa movement revealed that RLJ at various concentrations significantly (p < 0.05) negatively affected the progressive motility of harvested sperm cells from the caudal epididymides. This resulted in a concentration-dependent decrease, reducing from 85.27±1.8% (normal rats' progressive motility) to a range of 62.47±4.40–68.30±1.95%. In contrast, rats administered ULJ at 25%, 50%, and 75% concentrations recorded spermatozoa progressive motility of 79.73±2.86%, 73.98±3.49%, and 73.80±4.45%, respectively. These values were comparable (p > 0.05) with the normal rats' progressive motility (Table 3). The results of the spermatozoa live proportion in Wistar rats, as presented in Table 3, exhibited a direct relationship with the percentage of motile spermatozoa. The administration of RLJ had a negative effect (p < 0.05) on the livability of spermatozoa harvested from the caudal epididymides, while ULJ did not affect the live proportion. Ogbonnaya et al. – Comparative Effects of Ripe and Unripe Lime (Citrus aurantifolia) … 113 The spermatozoa concentration results showed gradual (p < 0.05) decreases in the number of sperm cells counted in each caudal epididymis of all the Wistar rats administered various concentrations of RLJ and in the rats administered 75% ULJ. However, the number of spermatozoa counted in the Wistar rats administered 25% (104.59±7.95 × 106/caudal epididymis) and 50% (84.42±15.90 × 106/caudal epididymis) ULJ were statistically comparable (p > 0.05) to the number counted in the control rats (111.19±7.37 × 106/caudal epididymis). Table 3. Effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa assessment of male wistar rats. Parameters Treatment group Colour Consistency pH Progressive motility (%) Spermatozoa livability (%) Spermatozoa concentration (×106/CE) 5 ml/kg D/W 1.00±0.00b 4.00±0.00 7.00±0.00a 85.27±1.8a 94.72±0.72a 111.19±7.37a 25% RLJ 1.00±0.00ab 3.33±0.33 6.99±0.00ab 68.30±1.95bc 82.47±1.38bc 64.33±3.84b 50% RLJ 1.66±0.33ab 3.33±0.67 6.99±0.00ab 64.67±6.64c 79.17±5.42bc 62.75±3.36b 75% RLJ 1.66±0.33ab 3.33±0.66 6.99±0.00ab 62.47±4.40c 75.51±4.55c 58.60±9.21b 25% ULJ 2.00±0.00a 3.66±0.00 6.99±0.00ab 79.73±2.86ab 87.54±2.96ab 104.69±7.95a 50% ULJ 2.00±0.00a 4.00±0.00 6.98±0.01b 73.96±3.49abc 84.99±3.75abc 84.42±15.90ab 75% ULJ 1.33±0.33ab 3.66±0.33 6.98±0.01b 73.80±4.45abc 84.77±3.21abc 77.54±5.00b Note: Values are presented as mean± S.E (standard error of mean). Different superscript letters along treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water. Effect of graded concentration of ripe and unripe lime juice extract on the spermatozoa morphology of Wister rats The results of spermatozoa morphology in the experimental male rats (Table 4) revealed that administering 50% and 75% of RLJ caused significant (p < 0.05) damage to the tail, head, and mid-piece of the sperm cells. Administration of ULJ at 75% also caused significant (p < 0.05) damage to the sperm cell head. However, the percentage of normal sperm cells in 25% RLJ concentration and 25%, 50%, and 75% ULJ were statistically comparable (p > 0.05) to the control rats. It is noteworthy to mention that 75% of RLJ caused the most significant (p < 0.05) damage to the cells, with the highest number of total abnormalities (4.71±0.50%) and the least (p < 0.05) number of normal cells (95.28±0.50%) compared to others. However, it's important to note that the total abnormal sperm cells obtained in 75% RLJ are less than the 25% concentration, which is considered to affect fertility. Table 4. Effect of graded concentration of ripe and unripe matured lime juice extract on spermatozoa morphology of male Wistar rats. Treatment group Tail abnormality Head abnormality Mid-piece abnormality Cytoplasmic droplets Total abnormal sperm cells Percentage normal sperm cells 5 ml/kg D/W 0.19±0.03de 0.27±0.02c 0.13±0.02c 1.16±0.05b 1.76±0.05c 98.23±0.05a 25% RLJ 0.27±0.02cd 0.26±0.04c 0.26±0.03bc 1.12±0.01b 1.91±0.05c 98.08±0.05a 50% RLJ 0.36±0.02b 1.25±0.03b 0.55±0.11b 1.12±0.00b 3.29±0.13b 96.71±0.13b 75% RLJ 0.44±0.03a 1.60±0.24a 1.41±0.26a 1.25±0.04a 4.71±0.50a 95.28±0.50c 25% ULJ 0.17±0.29e 0.23±0.01c 0.26±0.01bc 1.16±0.01b 1.83±0.02c 98.17±0.02a 50% ULJ 0.21±0.01de 0.31±0.04c 0.34±0.04bc 1.21±0.02ab 2.07±0.01c 97.92±0.00a 75% ULJ 0.33±0.01bc 0.32±0.02c 0.28±0.01bc 1.19±0.01ab 2.13±0.03c 97.87±0.03a Note: Values are presented as mean± S.E (standard error of mean). Different superscript letters along treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water. Effect of graded concentration of ripe and unripe matured lime juice extract on libido assessment of male Wistar rats The results regarding the effect of administering both RLJ and ULJ on the libido of male Wistar rats, as presented in (Table 5), indicated a concentration- dependent decrease in sex drive (libido) in both RLJ and ULJ administered groups compared to the high sex drive observed in the normal control Wistar rats. The reaction time outcomes revealed that it took an average of 7 minutes and 27 seconds, 5 minutes and 29 seconds, 6 minutes and 40 seconds, and 6 minutes and 49 seconds for the rats administered 25% RLJ, 25%, 50%, and 75% ULJ, respectively, to start grooming and sniffing the introduced female. In comparison, it took an average of 4 minutes and 26 seconds for those in the control group to complete the sexual behavioral reactions from general grooming to an attempt to mount. 114 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 109-119 Table 5. Effect of graded concentrations of ripe and unripe lime juice extract on libido of Wister rats. Treatment group Libido score Reaction Time (minutes) 5 ml/kg D/W 4.66±0.33a 4.26±0.45c 25% RLJ 1.66±0.26cd 7.27±0.64a 50% RLJ 1.00±0.00d 23.32±0.22b 75% RLJ 1.00±0.00d 24.47±0.53ab 25% ULJ 3.66±0.28b 5.29±0.86bc 50% ULJ 2.33±0.33c 6.40±0.48ab 75% ULJ 2.00±0.00c 6.49±0.72ab Note: Values are presented as Mean ± SEM (Standard Error of Mean). Different superscript letters across treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water Effect of graded concentration of ripe and unripe lime juice extract on body weight changes of Wister rats The body weight of the Wistar rats before the commencement of the lime juice administration (week 0) indicated that all the Wistar rats had the same body weight (p > 0.05) (Table 6). Furthermore, there was no significant (p > 0.05) difference in their body weight at week 1 during the administration of both RLJ and ULJ. However, by week 2, except for the average body weight of the rats administered a low concentration (25%) of RLJ, which weighed the same (p > 0.05) as the control rats, there was a significant and steady weight reduction across all the administered groups from week 2 to week 4. This was in contrast to the consistent (p < 0.05) body weight increase observed in the control rats during the same period. Table 6. Effect of graded concentration of ripe and unripe lime juice extract on the weight gain of Wister rats. Duration Treatment group Week 0 Week 1 Week 2 Week 3 Week 4 5 ml/kg D/W 217.00±17.32 228.50±8.74 245.00±9.65a 258.50±5.10a 264.50±20.10a 25% RLJ 216.25±10.76 223.00±6.79 218.25±23.26ab 213.50±4.97b 198.00±11.45b 50% RLJ 212.75±7.70 210.00±3.18 208.00±5.35b 193.25±5.26b 184.75±7.39bc 75% RLJ 214.25±10.47 210.50±3.52 210.00±3.18b 192.75±11.77b 159.75±6.28c 25% ULJ 211.50±9.04 210.75±4.92 192.25±6.77b 187.50±4.99b 173.25±11.85bc 50% ULJ 216.25±1.54 216.00±6.74 207.25±2.92b 192.75±11.77b 189.00±1.63bc 75% ULJ 216.75±3.68 219.00±7.12 210.25±1.70b 200.25±23.28b 196.25±6.01b Note: Values are presented as mean± S.E (standard error of mean). Different superscript letters along treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water. Effect of graded concentration of ripe and unripe lime juice extract on gonadosomatic index evaluation of Wister rats Table 7 displayed the relative weight of the testes in all the ULJ groups, and that of the 25% RLJ groups, which weighed the same (p > 0.05) as the size of the testes in the control group relative to their body weights. There was a significant (p < 0.05) reduction in the relative size of the testes in the rats administered 50% and 75% RLJ compared to the normal relative size of rat testes. However, the relative size of the prostate gland in the 75% RLJ administered rats was significantly (p < 0.05) reduced, indicating a smaller average prostate size compared to the control rats. Table 7. Effect of graded concentration of ripe and unripe lime juice extract on the Gonadosomatic index of Wister rats. Gonads Treatment group % Testes % Prostate % Seminal vesicle 5 ml/kg D/W 1.43±0.19a 0.16±0.03a 0.58±0.11a 25% RLJ 1.03±0.02abc 0.14±0.02ab 0.36±0.02b 50% RLJ 0.92±0.01c 0.07±0.01ab 0.21±0.06b 75% RLJ 0.94±0.02bc 0.05±0.04b 0.21±0.03b 25% ULJ 1.36±0.17ab 0.07±0.00ab 0.28±0.06b 50% ULJ 1.21±0.20abc 0.13±0.02ab 0.30±0.01b 75% ULJ 1.19±0.07abc 0.15±0.02a 0.28±0.07b Note: Values are presented as mean± S.E (standard error of mean). Different superscript letters along treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water. Ogbonnaya et al. – Comparative Effects of Ripe and Unripe Lime (Citrus aurantifolia) … 115 Effect of graded concentration of ripe and unripe lime juice extract on relative organ weight of Wister rats The results presented in Table 8 indicate that both RLJ and ULJ did not have a significant effect on the size of the liver, spleen, heart, kidneys, and lungs of the rats. The size of these visceral organs in the administered groups was statistically the same (p > 0.05) as the normal size of these organs in the control rats. This suggests that both RLJ and ULJ, at the various concentrations, did not cause any gross damage to the evaluated organs. Table 8. Effect of graded concentration of ripe and unripe lime juice extract on the relative organ weight of Wister rats. Visceral organs Treatment group Liver Spleen Heart Kidneys Lungs 5 ml/kg D/W 3.38±0.72 0.36±0.09 0.35±0.05 0.62±0.04 0.62±0.05 25% RLJ 3.51±0.46 0.47±0.13 0.34±0.02 0.65±0.12 0.77±0.07 50% RLJ 3.39±0.15 0.51±0.03 0.38±0.03 0.65±0.05 0.70±0.01 75% RLJ 2.93±0.09 0.44±0.02 0.33±0.02 0.48±0.01 0.58±0.04 25% ULJ 3.07±0.32 0.50±0.09 0.38±0.01 0.62±0.03 0.79±0.05 50% ULJ 3.48±0.41 0.45±0.10 0.33±0.03 0.54±0.04 0.68±0.03 75% ULJ 3.18±0.10 0.63±0.16 0.31±0.00 0.54±0.04 0.75±0.14 Note: Values are presented as mean± S.E (standard error of mean). Different superscript letters along treatment groups shows significant (p<0.05) differences. RLJ: Ripe lime juice; ULJ: Unripe lime juice; D/W: Distilled water. DISCUSSION The yield and concentration of RLJ and ULJ exhibited an inverse relationship. RLJ yielded 72.50% with a concentration of 0.08g/ml, while ULJ had a yield of 61.25% with a concentration of 0.1g/ml. This variation in both percentage yield and concentration gradient might have impacted the observed activity or effects of the two lime juices in this study. The chemical composition of RLJ includes essential components such as vitamins, minerals, phenolic compounds, saponins, cardiac glycosides, reducing sugars, flavonoids, and a minimal amount of steroids, which are recognized as crucial precursors to reproductive hormones (Enejoh et al., 2015; Navabi et al., 2018; Khan et al., 2018; Suntar et al., 2018; Saini et al., 2022). RLJ also contains a high level of tannins. Lime is known to have secondary metabolites like limonoids, phenylethylamine alkaloids, with p- synephrine being the most abundant (Adokoh et al., 2019). As indicated by Kim (2023), a medium-sized lime (67g) contains varying amounts of riboflavin, niacin, folates, phosphorus, magnesium, and trace amounts of iron, calcium, vitamin B6, and thiamine, along with a moderate amount of potassium and a substantial 22% of vitamin C. These phytoconstituents are scientifically believed to contribute to the physiological and pharmacological effects induced by lime juice on the body system (Jayaprakasha et al., 2006; Boshtam et al., 2011; Nallely et al., 2012; Akhtar, 2013; Saini et al., 2022). The alteration of physiological parameters, including rectal temperature, heart rate, weakness, behavioral pattern, skin fur, and appetite, used for toxicity evaluation, deviated from normal at the highest dose (5000 mg/kg) in both RLJ and ULJ. This deviation occurred shortly before their demise, within less than 1 hour post-administration, indicating an LD50 (lethal dose for 50% of the population) of 1581.138 mg/kg (<2000 mg/kg) for both lime juices. This suggests that both RLJ and ULJ have a narrow margin of safety. The findings of this study on LD50 align with the research conducted by Chijoke-Nwachukwu and Dede (2010), who reported an oral single dose LD50 of 1500 mg/kg for RLJ and 2000 mg/kg for lemon juice. Additionally, Oboma et al. (2020) and Saini et al. (2022) reported LD50 values for lime leaf extract in separate acute and subacute studies (1480 mg/kg, 1655 mg/kg, and 1532.64 mg/kg, with an average of 1555.88 mg/kg). This average did not significantly differ from the LD50 value obtained in the current study. The toxicity results imply that RLJ, with a 100% mortality rate, was more toxic to the mice than ULJ, even though RLJ is sourer than ULJ. This observation underscores the notion that ripeness does not necessarily correlate with safety in this context. The results obtained from the anti-fertility study revealed that ULJ at low to medium concentrations had a significant (p<0.05) effect on the color of the epididymal reserve in male Wistar rats, while RLJ did not exhibit any impact on the color. In Wistar rats, the normal color of semen is typically milky white, and changes to a creamy white color can indicate the influence of diet or treatment on semen color (Peters et al., 2008). There is a direct relationship between semen color and sperm concentration, with creamy white semen indicating a higher sperm count compared to milky white semen (Alkan et al., 2001; Peters et al., 2008). This observation aligns with the findings of the current study, where the rat group with creamy white epididymal reserve recorded a higher sperm count than those with milky white epididymal reserve. Similar correlations between semen color and sperm concentration have been reported in previous studies by Singh and Singh (2016), Ahmed et 116 Biology, Medicine, & Natural Product Chemistry 13 (1), 2024: 109-119 al. (2021), and Ezeigwe et al. (2022) in their fertility studies involving lemon fruits. However, Rahaju and Hanizar (2021) reported no direct relationship between semen color and sperm concentration in mice treated with a 500mg/kg dose of lime leaf extract. These findings suggest that ULJ, particularly at low to medium concentrations, may have a noticeable impact on the sperm count and epididymal reserve color in male Wistar rats, highlighting the importance of considering concentration levels in the evaluation of such effects. The observed interaction between higher concentrations of ULJ and spermatozoa stored in the caudal epididymides may have led to a decrease in pH, ultimately affecting sperm quality. Adokoh et al. (2019) reported a pH value of 6.67 in rat blood when ULJ was administered at a dose of 1000mg/kg for 58 days. This pH value is more acidic compared to the pH values of 6.98 obtained in the ULJ at 5% and 75% concentrations in the current study. The variation in pH values of ULJ in semen, as found in this study, could be attributed to differences in the buffering capacities of various body fluids or variations in dosage levels. The buffering capacity of a fluid refers to its ability to resist changes in pH when an acid or base is added. Differences in dosage levels might also influence the extent of the observed pH changes. It's worth noting that pH is a critical factor influencing the activity and function of sperm. Sperm function optimally within a specific pH range, and deviations from this range can adversely affect sperm quality and fertility. Therefore, the observed decrease in pH associated with higher concentrations of ULJ suggests a potential impact on sperm quality, emphasizing the importance of considering pH levels in the evaluation of reproductive effects. The findings from this study suggest that the administration of RLJ for 28 consecutive days led to a decrease in the progressive motility of spermatozoa. This result aligns with existing literature, including studies by Imade et al. (2005), Obidi et al. (2008), Sagay et al. (2009), Loizzo et al. (2012), Aprioku and Obianime (2014), Oboma et al. (2020), Ahmed et al. (2021), and Saini et al. (2022). These studies, as mentioned, have reported adverse effects on both female (such as reduction in the length of proestrus and estrus, increased frequency of metestrus, and changes in luteinizing hormone levels) and male (including decreased sperm motility, count, and viability) reproductive performance at higher dosages or concentrations of lime juice and Citrus aurantifolia leaf extracts administered to laboratory animals. While some studies, such as Singh and Singh (2016), have reported adverse effects of lemon and lime extracts on steroidogenic markers in the testes, induction of germ cell apoptosis, drastic decrease in epididymal sperm quality, and no effect on libido, others like Rahaju and Hanizar in 2021 reported improved sperm quality at 25% lemon leaf extract and negative effects at 75% concentration. The current study found that increasing the dose or concentration of lime juice adversely affected body fluids and reproductive performance, including the progressive motility and livability of sperm. The negative impact on the livability of spermatozoa corresponds with the reduction in their progressive motility. This observation is consistent with Bearden et al. (2004), who reported a high correlation between the proportion of live spermatozoa and visual estimates of progressively motile sperm cells. According to Anderson (2001), various external and internal factors may influence the production of semen and spermatozoa concentration, ultimately affecting male fertility. The study revealed that the administration of lime juice, particularly RLJ, for 28 days resulted in a low sperm count in Wistar rats. However, the reason why RLJ affected the spermatozoa indices evaluated more than ULJ remains unclear and warrants further investigation. From the study results, it is observed that the 75% concentration of RLJ caused significant abnormalities in the tail, head, and mid-piece of sperm, along with a higher percentage of cytoplasmic droplets compared to lower concentrations. The preservation of normal sperm morphology and integrity in the 25% RLJ, 25%, and 50% ULJ administered groups may be attributed to better antioxidant properties in both lime juices at lower concentrations. Regarding sexual drive, both RLJ and ULJ led to a decrease in sexual drive (libido), suggesting the presence of active ingredients that may have affected the release of interstitial cell-stimulating hormone and testosterone. The decrease in sexual drive was concentration- dependent and followed a similar trend as sperm concentration, indicating a direct relationship between libido and sperm concentration. However, there are variations in findings reported by Singh and Singh (2016), which showed no effect on libido with 1000 mg/kg of lime leaf extract, possibly due to differences in the plant parts used. Both RLJ and ULJ caused a significant reduction in body weights from week 2 to week 4. This reduction may be attributed to effects on metabolism, increased basal metabolic rate, suppressed appetite, induced lipolysis, or alteration in feed conversion efficiency. The observed reduction in prostate size in the 75% RLJ group is noteworthy, suggesting a potential effect on prostate health. The reduction in testicular size, particularly in the 50% and 75% RLJ concentrations, could be linked to ecobolic compounds in RLJ with estrogenic and spermicidal properties. These findings align with reports by Saini et al. (2022). While the study did not show significant enlargement, atrophy, or damage to major organs, it is important to note that changes in organ weights can be sensitive indicators of toxicity. The study suggests that RLJ and ULJ may have concentration-dependent effects on reproductive and physiological parameters in male Wistar rats, highlighting the need for further Ogbonnaya et al. – Comparative Effects of Ripe and Unripe Lime (Citrus aurantifolia) … 117 investigation into the underlying mechanisms and potential health implications. CONCLUSION This study, designed to investigate the effect of both RLJ and ULJ administration on the reproductive efficiency of male Wistar rats, revealed that ULJ is more concentrated than RLJ, although both recorded the same LD50 of 1581.138mg/kg, indicating a narrow safety margin. 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